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The Use of MasterTaq Polymerase for PCR with Humic Material-Contaminated ,,, DNA

PCR amplification. Two universal bacterial primers EUB008 and EUB1492 were used to amplify 16S rDNA genes from the extracted and purified
chromosomal DNA.11,12 PCR was performed with a Eppendorf Mastercycler gradient** as follows: 50 pmol of each primer, 2.5 mol of each deoxyribonucleoside triphosphate, 300 g bovine serum albumin (BSA), 1x reaction buffer, 1x TaqMaster PCR enhancer and 1U MasterTaq DNA Polymerase were adjusted to a final volume of 100 l with sterile water. From 80 ng to 500 ng of template DNA was added to the reaction mixture after preheating to 70C to avoid nonspecific annealing of the primers to nontarget DNA. The cycles used were:

1 cycle at 70C for 1 min, followed by 38 cycles at 95C for 1 min, 40C for 1 min, 72C for 3 min and 1 final cycle at 95C for 1 min, 40C for 1 min and at 72C for 10 min. In parallel, the same experiment was performed using a conventional Taq polymerase, which was routinely used in our lab and considered to be of maximal suitability for PCR. DNA extracted from E. coli was used as a positive control.

The use of high concentrations of BSA in the hot-start PCR was shown to decrease the sensitivity of the reaction against components in the soil extracts and to make PCR product formation reproducible.


Fig. 1: Use of MasterTaq and conventional Taq polymerase for the amplification of 16S rDNA genes from humic-acid contaminated DNA extracted from a marine sediment. PCR products were analyzed on a 1% agarose gel.

Lanes 1 and 4: E. coli-DNA (= positive controls)
Lanes 2 and 5: negative control (H2O)
Lanes 3 and 6: community DNA, extracted from a marine sediment



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